In vitro activities of ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam and other comparators against Pseudomonas aeruginosa isolates with discrepant resistance to carbapenems: Data from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program, 2012-2021.

OBJECTIVES: This study aimed to investigate the in vitro susceptibility and ß-lactamase-encoding genes of Pseudomonas aeruginosa (P. aeruginosa) isolates with discrepant resistance to various carbapenems. METHODS: Data on P. aeruginosa isolates were obtained from the Antimicrobial Testing Leadership and Surveillance program from 2012-2021. Minimum inhibitory concentrations of P. aeruginosa isolates were determined using the broth microdilution method. ß-lactamase-encoding genes were identified using multiplex polymerase chain reaction assays. RESULTS: Among the P. aeruginosa isolates that were tested, the percentages of isolates resistant to imipenem, meropenem and doripenem were 26.9% (14 447 of 53 617), 20.5% (14 098 of 68 897) and 17.5% (3660 of 20 946), respectively. Imipenem-resistant P. aeruginosa isolates were more susceptible to all tested antimicrobial agents (except colistin) than the meropenem-resistant or doripenem-resistant P. aeruginosa isolates. Carbapenemase genes were detected in 14.3% (2020 of 14 098) of meropenem-resistant P. aeruginosa isolates. Imipenem-resistant meropenem-susceptible P. aeruginosa isolates had higher susceptibility profiles, fewer carbapenemase genes (0.3% [five of 1858] vs. 4.1% [10 of 242]; P < 0.05) and a lower risk of being classified as multidrug-resistant than the imipenem-susceptible meropenem-resistant isolates (16.1% [299 of 1858] vs. 73.6% [178 of 242]; P < 0.05). Among all ß-lactam combination agents, ceftazidime-avibactam and ceftolozane-tazobactam had higher susceptibility rates than meropenem-vaborbactam for meropenem-resistant P. aeruginosa (61.8% and 55.5% vs. 30.2%; P < 0.05). CONCLUSION: Discrepancy in the resistance of different P. aeruginosa isolates to various carbapenems suggests their different underlying resistance mechanisms. These findings can be useful for effective resistance trend monitoring and accurate antimicrobial treatment in the future.

Auranofin promotes antibacterial effect of doripenem against carbapenem-resistant Acinetobacter baumannii.

AIMS: This study was performed to identify the potential for repurposing auranofin as an antibiotic adjuvant against carbapenemase-producing Acinetobacter baumannii. METHODS AND RESULTS: The clinically isolated A. baumannii strains used in this study were all resistant to carbapenems and harboured the blaOXA-23 gene. The synergistic effect of auranofin and doripenem against carbapenemase-producing A. baumannii was confirmed through checkerboard and growth kinetic analyses. This study also demonstrated the inhibitory effects of auranofin against A. baumannii biofilms. The anti-biofilm effects of auranofin were visualized by confocal laser scanning microscopy (CLSM). Furthermore, auranofin inhibited motility, one of the virulence factors. Additionally, the changes in the expression of carbapenemase-, biofilm- and efflux pump-related genes induced by auranofin were confirmed via quantitative polymerase chain reaction (qPCR). CONCLUSIONS: Our results demonstrated that auranofin has an antibacterial effect with doripenem and an inhibitory effect on several factors related to carbapenem resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that auranofin is a promising antibiotic adjuvant that can be used to prevent antibiotic resistance in carbapenem-resistant A. baumannii.

Arginine Modulates Carbapenem Deactivation by OXA-24/40 in Acinetobacter baumannii.

The resistance of Gram-negative bacteria to ß-lactam antibiotics stems mainly from ß-lactamase proteins that hydrolytically deactivate the ß-lactams. Of particular concern are the ß-lactamases that can deactivate a class of ß-lactams known as carbapenems. Carbapenems are among the few anti-infectives that can treat multi-drug resistant bacterial infections. Revealing the mechanisms of their deactivation by ß-lactamases is a necessary step for preserving their therapeutic value. Here, we present NMR investigations of OXA-24/40, a carbapenem-hydrolyzing Class D ß-lactamase (CHDL) expressed in the gram-negative pathogen, Acinetobacter baumannii. Using rapid data acquisition methods, we were able to study the "real-time" deactivation of the carbapenem known as doripenem by OXA-24/40. Our results indicate that OXA-24/40 has two deactivation mechanisms: canonical hydrolytic cleavage, and a distinct mechanism that produces a ß-lactone product that has weak affinity for the OXA-24/40 active site. The mechanisms issue from distinct active site environments poised either for hydrolysis or ß-lactone formation. Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling ß-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.

Resistência aos antimicrobianos e diversidade genética de variantes fenotípicas de Pseudomonas aeruginosa não sensíveis aos carbapenêmicos recuperadas de pacientes com fibrose cística com infecção pulmonar crônica: uma análise temporal / Antimicrobial resistance and genetic diversity of phenotypic variants of carbapenem non susceptible Pseudomonas aeruginosa recovered from patients with cystic fibrosis with chronic lung infection: a temporal analysis

Pseudomonas aeruginosa, bactéria ubíqua e versátil, pode se comportar como um patógeno oportunista, com ampla capacidade adaptativa, por múltiplos fatores de virulência e resistência. Como agente patogênico nas infecções pulmonares em pacientes com fibrose cística (FC), é motivo de prognóstico ruim, aumento de hospitalizações e altas taxas de morbimortalidade, sendo quase impossível a sua erradicação, ao evoluírem para a cronicidade. Globalmente, é notável o aumento nos índices de amostras não sensíveis aos carbapenêmicos e a múltiplos antimicrobianos, essenciais à terapêutica. Assim, avaliamos temporalmente a susceptibilidade aos antimicrobianos e a presença de amostras hipermutáveis (HPM) em P. aeruginosa de diferentes morfotipos, não sensíveis aos carbapenêmicos (PANSC), obtidas de pacientes FC com infecção pulmonar crônica, acompanhados em dois centros de referência no Rio de Janeiro. De 2007 a 2016, a análise retrospectiva, através dos resultados obtidos no teste de disco-difusão (TDD), permitiu selecionar amostras de PANSC incluídas neste trabalho. Usando os resultados obtidos no TDD, foi definida a susceptibilidade a outros antimicrobianos, bem como os fenótipos de resistência, multi-(MDR), extensivo-(XDR) e pandroga resistentes (PDR). Adicionalmente, determinou-se a concentração inibitória mínima (CIM) para imipenem (IPM), meropenem (MEM), doripenem (DOR) e polimixina (POL). Através de teste fenotípico, foi calculada a frequência de mutação espontânea e as amostras hipermutáveis foram caracterizadas. O sequenciamento de genoma total (SGT) foi realizado em seis amostras de diferentes morfotipos, incluindo uma variante fenotípica rara, a small colony variant (SCV). Essas amostras foram recuperadas em dois episódios de exacerbação do paciente. Foram investigadas a clonalidade, resistência a antimicrobianos e virulência. Das 143 amostras, de 18 pacientes (9 pediátricos e 9 adultos), os resultados do TDD apontaram taxas de não susceptibilidade superiores a 44% para gentamicina, amicacina, tobramicina e ciprofloxacina, e maiores de 30 % para POL. Pela determinação da CIM, quase a totalidade (96%) das amostras foram não sensíveis a IMP, seguidos de 56% para MEM e 44% para DOR. Analisando-se a distribuição dos valores da CIM50 e CIM90 nos dois grupos de pacientes, os valores para IMP foram maiores entre as amostras dos pacientes pediátricos, equivalendo a 32 µg/mL e 64 µg/mL, respectivamente. Cerca de 25%, 37% e 6% eram MDR, XDR e PDR, respectivamente. Aproximadamente 12% eram HPM, e mais da metade destas foram XDR. Após o SGT, as seis amostras, recuperadas do caso clínico foram classificadas em um novo sequence type (ST2744), com a presença de genes de resistência adquiridos blaPAO, blaOXA-50, aph(3')-Iib, fosA, catB7 e crpP, apresentando mutações em genes codificadores de porinas e bombas de efluxo. Entretanto, não foram observados marcadores genéticos clássicos exclusivos para os fenótipos SCV e HPM. Este é o primeiro relato de P. aeruginosa SCV na FC, no Brasil. A vigilância epidemiológica de P. aeruginosa é crucial para a conduta terapêutica, bem como para o sucesso da resposta do paciente e erradicação da infecção pulmonar, justificando o uso de técnicas fenotípicas e moleculares na detecção dos mecanismos de resistência e virulência desse microrganismo na FC.

Pseudomonas aeruginosa, a ubiquitous and versatile bacterium, can behave as an opportunistic pathogen, with strong adaptive capacity, due to multiple virulence and resistance factors. As a pulmonary infection pathogen in patients with cystic fibrosis (CF), it is related with poor prognosis, increased hospitalizations and high rates of morbidity and mortality, and the eradication is almost impossible, especially after chronicity. The increase rates of isolates non-susceptible to carbapenem and multiple antimicrobials, essentials to therapy, have been observed worldwide. Therefore, we assessed the antimicrobial susceptibility and the presence of hypermutability (HPM) in non-susceptible to carbapenem P. aeruginosa (PANSC) isolates from different morphotypes, obtained from CF patients with chronic pulmonary infection, followed at two reference centers in Rio de Janeiro. Using the results obtained by disk-diffusion test (DDT) between 2007 to 2016, we select 143 PANSC and susceptibility to other antimicrobials was defined, as well as the resistance phenotypes, multi- (MDR), extensive- (XDR) and pandrug resistant (PDR). Additionally, the minimum inhibitory concentration (MIC) for imipenem (IPM), meropenem (MEM), doripenem (DOR) and polymyxin (POL) was determined. Hypermutable isolates were characterized by determination of mutation frequency. Whole genome sequencing (WGS) was performed in six morphotypes isolates, including the small colony variant (SCV), a rare variant phenotype. These isolates were recovered in two exacerbation episodes. Clonality, antimicrobial resistance and virulence were investigated. Of the total (143 isolates) isolated from 18 patients (9 pediatric and 9 adults), non-susceptibility rates above than 44% for gentamicin, amikacin, tobramycin and ciprofloxacin, and more than 30% for POL were observed. Almost all (96%) of the isolates were non-susceptible to IPM by MIC determination, followed by 56% for MEM and 44% for DOR. MIC50 (32 µg/mL) and MIC90 (64 µg/mL) rates for IPM were higher among pediatric patient isolates and 25%, 37% and 6% were MDR, XDR and PDR, respectively. 12% of all isolates were classified as HPM and more than half were categorized as XDR. Using WGS, the six isolates recovered from the clinical case, were identified as a new sequence type (ST2744). Acquired resistance genes blaPAO, blaOXA-50, aph (3')-Iib, fosA, catB7 and crpP and mutations in encoding genes for porins and efflux pumps, was annotated. None exclusive classic genetic markers related to SCV and HPM phenotypes were not observed. This is the first Brazilian report of P. aeruginosa SCV in CF. Our results highlight the importance of epidemiological surveillance in P. aeruginosa. The application of phenotypic and molecular techniques to investigate resistance and virulence mechanisms, can contribute to therapeutic success in CF.

A method to quantify viable carbapenem resistant gram-negative bacteria in treated and untreated wastewater.

Carbapenem resistance, particularly in Enterobacteriaceae, is an urgent threat to public health worldwide. Wastewater treatment plants are a critical control point for the spread of antimicrobial resistance into the environment yet, due in part to the lack of appropriate methods, the occurrence, identification and removal of carbapenem resistant bacteria has not been well characterized in wastewater matrices. This project was designed to provide a method for quantification of viable carbapenem resistant (CR) gram-negative bacteria (GNB) in raw sewage and treated wastewater effluents. A two-step procedure using membrane filtration and selective media supplemented with each of four carbapenems (doripenem, meropenem, imipenem, and ertapenem) was established for the quantification of CR GNB in wastewater matrices. Carbapenemase production was also assessed on individual bacterial colonies using two separate methods. Vitek®2 antimicrobial susceptibility test and disk diffusion assays were used to verify results from the supplemented media test and provide taxonomic identification. Treated and untreated wastewater samples from secondary and tertiary-stage wastewater treatment plants were analyzed for CR bacteria using the supplemented media procedure. Over 98% of all isolates selected from the carbapenem-supplemented media were verified as CR GNB. Carbapenemase production was observed in 80% of these isolates and 88% were multidrug resistant. All Enterobacteriaceae isolates from the supplemented media were verified as CR and 97% tested positive for carbapenemase production. The highest concentrations of CR GNB in wastewater were observed using the ertapenem-supplemented media. Doripenem-supplemented media showed the greatest specificity and selectivity for carbapenemase-producing CRE. Overall, the cumulative CR GNB in wastewater were reduced by approximately three- and five-log10 by the secondary and tertiary-stage WWTPs, respectively. This study establishes a method for characterization of viable CR GNB in wastewater matrices and demonstrates that current wastewater treatment technologies effectively reduce CR bacteria, including CRE, in sewage.

Antimicrobial susceptibility and prevalence of resistance genes in Bacteroides fragilis isolated from blood culture bottles in two tertiary care hospitals in Japan.

The in vitro susceptibilities of Bacteroides fragilis to antimicrobial agents, especially to carbapenem, are a major concern in the treatment of patients with bloodstream infections. In this study, 50 isolates of B. fragilis were obtained from positive blood bottles from 2014 to 2019 in Saitama, Japan. Their susceptibility to ampicillin/sulbactam was reduced to 70.0% compared with a previous report, whereas they were still sufficiently susceptible to piperacillin/tazobactam (94.0%). Five cfiA-positive isolates (5/50, 10.0%) were identified that were resistant to doripenem and meropenem, and two of them carried an insertion sequence located upstream of the cfiA-coding region. In particular, imipenem should be considered as a first-line carbapenem for the empirical treatment of B. fragilis infection because only insertion sequence and cfiA double-positive strains showed resistance to imipenem. Thirty-six percent of the isolates had a reduced minimum inhibitory concentration for moxifloxacin. In addition, metronidazole should still be considered as an active agent for B. fragilis because all isolates were susceptible to this antibiotic and the prevalence of the nim gene was low in Japan.

Synergistic activities of ceftazidime-avibactam in combination with different antibiotics against colistin-nonsusceptible clinical strains of Pseudomonas aeruginosa.

Background: This study aims to analyse the effect of ceftazidime-avibactam plus various antibiotics against multidrug-resistant (MDR) Pseudomonas aeruginosa isolated from Intensive Care Units.Methods: 40 non-duplicate P. aeruginosa isolates were screened for their MICs of ceftazidime, ceftazidime-avibactam, colistin, levofloxacin, doripenem and tobramycin. MICs were determined by the broth microdilution method. The in vitro bactericidal activities of ceftazidime-avibactam compared to studied antibiotics were also determined by time-kill curve assays both at 1xMIC and at 4xMIC against carbapenemase-producing or -not producing six colistin-nonsusceptible MDR clinical strains of P.aeruginosa. Additionally, synergistic interactions were investigated by the time-kill curve assay.Results: The MIC90 values for ceftazidime, ceftazidime-avibactam, colistin, levofloxacin, doripenem and tobramycin against MDR P. aeruginosa isolates were found to be >256, 64, 8, 64, 128, and >256 mg/L, respectively. The minimum bactericidal concentration90 values for those antibiotics were also >256, 64, 16, 128, 256, and >256 mg/L, respectively. While doripenem, tobramycin and levofloxacin were bactericidal (>3 log10 killing) against the 2/6, 3/6 and 1/6 P. aeruginosa isolates at 4xMIC concentrations, respectively, levofloxacin and tobramycin were bactericidal against only one isolate (1/6) at 1xMIC concentrations at 24 h. The synergistic interactions of these antimicrobial agents were also achieved with ceftazidime/avibactam + colistin (4/6), ceftazidime/avibactam + tobramycin (3/6), and ceftazidime/avibactam + levofloxacin (3/6) combinations. No antagonism was observed against studied P. aeruginosa strains.Conclusions: The findings of this study suggest that ceftazidime/avibactam with colistin, or tobramycin, were effective against colistin-nonsusceptible strains. This combination therapy could be an alternative antibiotic therapy for resistant P. aeruginosa strains.

Evaluation of the synergy of ceftazidime/avibactam in combination with colistin, doripenem, levofloxacin, tigecycline, and tobramycin against OXA-48 producing Enterobacterales.

This study aims to analyze the effect of ceftazidime/avibactam plus various antibiotics against OXA-48-producing Enterobacterales isolated from Intensive Care Units. Seventy-four non-duplicate OXA-48-producing Enterobacterales isolates were screened for their MICs by the microbroth dilution method. The in-vitro bactericidal and synergistic activities of ceftazidime/avibactam alone or in combination with other antibiotics were determined by time-kill curve assays. According to our results, colistin was the most active drug with higher susceptibility rates in the strains. Colistin, levofloxacin, tobramycin, and doripenem showed bactericidal effects against different isolates. The best synergistic interactions were achieved with ceftazidime/avibactam + colistin, ceftazidime/avibactam + tobramycin, and ceftazidime/avibactam + tigecycline against studied strains used at 1xMIC concentrations at 24 h. No antagonism was observed against studied OXA-48-producing Enterobacterales strains.The findings of this study suggest that ceftazidime/avibactam plus colistin, tobramycin, or tigecycline were more effective against OXA-48-producing Enterobacterales strains. This combination therapy could be an alternative antibiotic therapy for carbapenemase-producing Enterobacterales strains.

An Integrated Dialysis Pharmacometric (IDP) Model to Evaluate the Pharmacokinetics in Patients Undergoing Renal Replacement Therapy.

PURPOSE: Clearance via renal replacement therapy (RRT) can significantly alter the pharmacokinetic profile of drugs. The aim of this study was (i) to improve the use of clinical trial data and (ii) to provide a model that allows quantification of all aspects of drug elimination via RRT including adsorption to dialysis membranes and/or degradation of the drug in the dialysate. METHODS: An integrated dialysis pharmacometric (IDP) model was developed to simultaneously incorporate all available RRT information. The sensitivity, accuracy and precision of the IDP model was compared to conventional approaches in clinical trial simulations and applied to clinical datasets of teicoplanin and doripenem. RESULTS: The IDP model was more accurate, precise and sensitive than conventional plasma-concentration-based approaches when estimating the clearanceRRT (relative bias <1%). In contrast to conventional approaches, adsorption and degradation were quantifiable using the IDP model (relative bias: -1.1% and - 1.9%, respectively). Applied to clinical data, clearanceRRT, drug degradation (effluent-half-lifedoripenem: 13.5 h-1) and adsorption (polysulphone adsorption capacityteicoplanin: 31.2 mg) were assessed. CONCLUSION: The IDP model allows accurate, precise and sensitive characterization of clearanceRRT, adsorption and degradation. Successful quantification of all aspects of clearanceRRT in clinical data demonstrated the benefit of the IDP model as compared to conventional approaches.

Pseudomonas Aeruginosa as the Main Causative Agent of Osteomyelitis and its Susceptibility to Antibiotics.

Surgical activity is increasing in the treatment of many types of fractures, the use of various metal structures, and the potential for infection with the development of osteomyelitis accordingly increases. The urgency of the problem is due to the fact that this disease is the most expensive medical problem, especially when it comes to prosthetics of large joints, with socially significant losses and the occurrence of disability in patients of working age, it requires long-term treatment. The aim of this study was to study one of the most complex pathogens of life-threatening infections due to its high virulence and ability to adapt to changing environmental conditions, in particular the action of antibacterial drugs and the study of its sensitivity to certain groups of antimicrobial drugs. The results of the study showed that over the three years of observation in 2017-2019, the average value of Pseudomonas aeruginosa in the amount of 10.8% was established in the etiology of osteomyelitis. The revealed sensitivity of Pseudomonas aeruginosa is preserved to fluoroquinolone - levofloxanin, aminoglycosides-amikacin, gentamicin, carbapenems - meropenem, doripenem. All of the above drugs can be used as empirical therapy. During this period, a significant decrease in sensitivity was found, which reached in 2019 for cefepime - 51.9%, pefloxacin - 55.8%, ertapenem - 59.7%. The success of the treatment of this pathology directly depends on the timely microbiological diagnosis and the choice of patient treatment tactics with the appointment of effective antibacterial therapy, with an adequate exposure of antibiotic.

In vitro and in vivo assessment of the antibacterial activity of colistin alone and in combination with other antibiotics against Acinetobacter baumannii and Escherichia coli.

OBJECTIVES: Limited therapeutic options exist for treating severe infections caused by multidrug-resistant (MDR) and extensively drug-resistant Gram-negative bacteria (GNB). In this study, the activity of colistin (COL) as monotherapy and in combination with other antibiotics against Acinetobacter baumannii in vitro was investigated. In addition, the efficacy of intravenous colistimethate sodium (CMS) was evaluated in a murine model of urinary tract infection (UTI) induced by MDR Escherichia coli. METHODS: Minimum inhibitory concentration (MIC), Monte Carlo simulation, fractional inhibitory concentration index (FICI), time-kill study and erythrocyte lysis assay were applied to evaluate the effect and cytotoxicity of COL, meropenem, imipenem, doripenem (DOR) and sulbactam alone and in combination. For the in vivo experiment, determination of the bacterial burden and histopathological examination were performed to evaluate the efficacy of CMS against UTI. RESULTS: Of 106 A. baumannii isolates, 104 (98.1%) were susceptible to COL. In the chequerboard assay, COL + DOR showed the highest rate of synergism (60%). No antagonism or cytotoxicity was observed. All COL-based combinations were able to inhibit or slow bacterial re-growth in a time-kill assay. In an in vivo activity study, intravenous CMS reduced not only the bacterial load but also inflammation and maintained structural integrity of infected bladders and kidneys. CONCLUSION: The effectiveness of COL alone in vitro and in vivo suggested that intravenous CMS will be an effective and available therapeutic strategy for UTI due to MDR-GNB. In-depth in vitro tests demonstrated that COL + DOR could be an attractive option, especially when the COL MIC is ≥1 µg/mL.

Structural Insights into the Mechanism of Carbapenemase Activity of the OXA-48 ß-Lactamase.

Carbapenem-hydrolyzing class D carbapenemases (CHDLs) are enzymes that produce resistance to the last-resort carbapenem antibiotics, severely compromising the available therapeutic options for the treatment of life-threatening infections. A broad variety of CHDLs, including OXA-23, OXA-24/40, and OXA-58, circulate in Acinetobacter baumannii, while the OXA-48 CHDL is predominant in Enterobacteriaceae Extensive structural studies of A. baumannii enzymes have provided important information regarding their interactions with carbapenems and significantly contributed to the understanding of the mechanism of their carbapenemase activity. However, the interactions between carbapenems and OXA-48 have not yet been elucidated. We determined the X-ray crystal structures of the acyl-enzyme complexes of OXA-48 with four carbapenems, imipenem, meropenem, ertapenem, and doripenem, and compared them with those of known carbapenem complexes of A. baumannii CHDLs. In the A. baumannii enzymes, acylation by carbapenems triggers significant displacement of one of two conserved hydrophobic surface residues, resulting in the formation of a channel for entry of the deacylating water into the active site. We show that such a channel preexists in apo-OXA-48 and that only minor displacement of the conserved hydrophobic surface residues occurs upon the formation of OXA-48 acyl-enzyme intermediates. We also demonstrate that the extensive hydrophobic interactions that occur between a conserved hydrophobic bridge of the A. baumannii CHDLs and the carbapenem tails are lost in OXA-48 in the absence of an equivalent bridge structure. These data highlight significant differences between the interactions of carbapenems with OXA-48 and those with A. baumannii enzymes and provide important insights into the mechanism of carbapenemase activity of the major Enterobacteriaceae CHDL, OXA-48.

Synergistic effect of doripenem in combination with cefoxitin and tetracycline in inhibiting NDM-1 producing bacteria.

Aim: To propose newer combinations of antibiotics effective against NDM-1-producing bacterial strains. Materials & methods: Antibiotics combinations were tested by checkerboard assay. NDM-1 protein/enzyme was expressed and purified to perform enzyme kinetics, circular dichroism and fluorescence spectroscopic studies. Results: Doripenem-cefoxitin combination and doripenem-tetracycline combination showed synergistic effect toward NDM-1-producing strains. The catalytic efficiency of NDM-1 enzyme was decreased drastically by 96.6% upon doripenem-cefoxitin treatment and by 35.54% after doripenem-tetracycline treatment. Conformational changes were observed in NDM-1 upon combination treatment. Conclusion: NDM-1-producing bacterial strains show resistance to multiple antibiotics but the combination of doripenem-cefoxitin and doripenem-tetracycline are effective against them. The combination of a carbapenem and cephamycin antibiotic is proposed for future treatment options against bacteria-producing NDM-1.

lptG contributes to changes in membrane permeability and the emergence of multidrug hypersusceptibility in a cystic fibrosis isolate of Pseudomonas aeruginosa.

PURPOSE: In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34. METHODS: PA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid-log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem-resistant colonies hypersusceptible to non-carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT-rtPCR and OprD production by SDS-PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem-resistant clinical isolates hypersusceptible to non-carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ-Emerald stain. RESULTS: Isogenic mutants showed 4- to 8-fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4-fold MIC decrease) to aminoglycosides, fluoroquinolones, and non-carbapenem ß-lactams. Expression of ampC or mex-opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non-synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics. CONCLUSION: Evaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility.

Highly Synergistic Effects of Melittin with Conventional Antibiotics Against Multidrug-Resistant Isolates of Acinetobacter baumannii and Pseudomonas aeruginosa.

BACKGROUND: Morbidity and mortality due to multidrug-resistant (MDR) bacteria are of great concern in burn patients. In this critical condition, synergism between antimicrobial peptides and conventional antibiotics would be a promising strategy. Accordingly, this study aimed to determine the therapeutic value of melittin as a natural peptide by examining its synergistic effect with conventional antibiotics against MDR isolates of Acinetobacter baumannii and Pseudomonas aeruginosa. MATERIALS AND METHODS: Fifteen clinical isolates for each kind of bacteria were collected from burn patients. Antibiotic susceptibility of all isolates was evaluated by disk diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration for melittin, colistin, doripenem, doxycycline, and ceftazidime were also examined. Fractional inhibitory concentration (FIC) of melittin in combination with the antibiotics was determined for six MDR isolates. The cytotoxicity of melittin in combination with the antibiotics was examined on a normal human cell line. RESULTS: The geometric means of MIC (GMMIC) for melittin and doripenem after combination were reduced to 61.5- and 51.5-fold, respectively, against MDR A. baumannii isolates. These reductions for melittin-doripenem and melittin-ceftazidime against MDR P. aeruginosa isolates were (63.5 and 58)-fold and (16 and 11)-fold, respectively. FIC for melittin-doripenem against A. baumannii and FIC for melittin-doripenem and melittin-ceftazidime against P. aeruginosa strains were ≤0.5. This issue caused a decrease of up to 104-, 68-, and 17-fold, respectively, in the cytotoxicity of melittin. CONCLUSION: In conclusion, the synergism of melittin at its nontoxic dose with doripenem and ceftazidime could be of great therapeutic value as a topical drug against burn infections caused by MDR bacteria.

Role of the Hydrophobic Bridge in the Carbapenemase Activity of Class D ß-Lactamases.

Class D carbapenemases are enzymes of the utmost clinical importance due to their ability to confer resistance to the last-resort carbapenem antibiotics. We investigated the role of the conserved hydrophobic bridge in the carbapenemase activity of OXA-23, the major carbapenemase of the important pathogen Acinetobacter baumannii We show that substitution of the bridge residue Phe110 affects resistance to meropenem and doripenem and has little effect on MICs of imipenem. The opposite effect was observed upon substitution of the other bridge residue Met221. Complete disruption of the bridge by the F110A/M221A substitution resulted in a significant loss of affinity for doripenem and meropenem and to a lesser extent for imipenem, which is reflected in the reduced MICs of these antibiotics. In the wild-type OXA-23, the pyrrolidine ring of the meropenem tail forms a hydrophobic interaction with Phe110 of the bridge. Similar interactions would ensue with ring-containing doripenem but not with imipenem, which lacks this ring. Our structural studies showed that this interaction with the meropenem tail is missing in the F110A/M221A mutant. These data explain why disruption of the interaction between the enzyme and the carbapenem substrate impacts the affinity and MICs of meropenem and doripenem to a larger degree than those of imipenem. Our structures also show that the bridge directs the acylated carbapenem into a specific tautomeric conformation. However, it is not this conformation but rather the stabilizing interaction between the tail of the antibiotic and the hydrophobic bridge that contributes to the carbapenemase activity of class D ß-lactamases.

Preparation and Characterization of Doripenem-Loaded Microparticles for Pulmonary Delivery.

Background: Pneumonia is a bacterial lower respiratory tract infection that has a high morbidity rate. The gram-negative pathogen Pseudomonas aeruginosa is a significant cause of nosocomial infections and ventilator-associated pneumonias and is mainly treated by carbapenems. Doripenem is a carbapenem drug, which has a broad-spectrum antibacterial activity. The aim of this study was to develop doripenem-loaded chitosan microparticles for pulmonary administration to provide more efficient treatment for pneumonia. Methods: Ionotropic gelation and the spray-drying method were used to obtain doripenem-loaded chitosan microparticles with different lactose, trehalose, and L-leucine concentrations. Physicochemical characteristics, in vitro drug release properties, and aerodynamics properties were investigated and in vitro antimicrobial susceptibility tests of the formulations were performed. Assessment of aerodynamic properties of the powders, including Mass Median Aerodynamic Diameter, size distribution, and fine particle fraction (FPF), were performed using a Next Generation Impactor. Cytotoxicity of the fabricated microparticles was assessed using the Calu-3 cell airway epithelial cell line. Results: Optimum microparticles were produced using a combination of ionotropic gelation and spray-drying methods. Spray-dried microparticle production yield was relatively high (74.03% ± 3.88% to 98.23% ± 1.70%). Lactose, trehalose, and L-leucine were added to the formulation to prevent aggregation produced by the ionotropic gelation spray-drying method. Each formulation's encapsulation efficiency was above 78.98% ± 2.37%. The doripenem-loaded microparticle mean diameter ranged from 3.8 ± 0.110 to 6.9 ± 0.090 µm. Microparticles with 20% (w/w) L-leucine had the highest FPF ratio indicating the best aerosolization properties of the formulations. The efficacy of the formulations as an antibacterial agent was increased by forming doripenem-loaded microparticles compared to blank microparticles. P. aeruginosa showed the same susceptibility to all doripenem-loaded microparticle formulations. Cell viability of microparticles was between 70% ± 0.08% and 90% ± 0.04% at 0.5 and 10 mg/mL concentration, respectively. Conclusions: Doripenem-loaded microparticles, produced using a combination of ionotropic gelation and spray-drying methods, are suitable for pulmonary drug delivery based on their particles size, zeta potential, cytotoxicity and high production yield. To our knowledge, this is the first study that microparticles containing doripenem were produced and characterized.

Interpreting carbapenem susceptibility testing results for Pseudomonas aeruginosa.

OBJECTIVES: Carbapenems are among the most powerful antipseudomonal agents. Limited data is available on drug susceptibility testing by routine methods (disc diffusion and Etest) for meropenem and doripenem. We aimed to compare the in vitro activity of imipenem, meropenem, and doripenem against Pseudomonas aeruginosa. METHODS: A total of 311 P. aeruginosa strains isolated from respiratory specimens in 170 patients who developed ventilator-associated pneumonia in two intensive care units were collected over a period of 31 months. The susceptibility of these isolates to imipenem, meropenem, and doripenem were determined by Etest and disc diffusion method. RESULTS: Considering either all isolates or only the first isolates recovered per patient (311 and 170 respectively), the susceptibility rate for doripenem was higher than that for meropenem and imipenem. When MICs determined by Etest were converted into interpretative categories (S, I, R) using French (CA-SFM) guidelines, a poor correlation was observed for meropenem and doripenem. The percentages of correlation with the disc diffusion method were 90.6% and 89.7% for imipenem, 80.5% and 82.6% for meropenem, and 80.5% and 73.3% for doripenem, for the first isolates and all isolates, respectively. The rate of minor errors was as high as 17.7% and 16.1% for meropenem and 17.7% and 25.7% for doripenem for the first isolates and all isolates, respectively. CONCLUSION: The accuracy of disc diffusion using CA-SFM guidelines appears unsatisfactory for all three carbapenems justifying guideline update for P. aeruginosa and carbapenems.

Intereactions between doripenem and clavulanate ­ Application of minimal inhibitory concentration analysis and cytometry flow for bactericidal studies

Background: In view of the current low efficacy of bacterial infection treatment the common trend towards searching for antibiotic systems exhibiting synergistic action is well justified. Among carbapenem analogues a particularly interesting option is provided by combinations of clavulanic acid with meropenem, which have proven to be especially effective. Results: Determination of the minimal inhibitory concentration (MIC) along with the method based on flow cytometry constitutes an important tool in the identification of bacterial sensitivity to active substances. Within this study the inhibitory effect of doripenem, clavulanic acid and the doripenem-clavulanate acid system was analyzed in relation to such bacteria as Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Clostridium butyricum and Clostridium pasteurianum, Acinetobacter baumannii, Enterobacter aerogenes. The lowest MIC, amounting to 0.03 µg/mL, was observed for the doripenem-clavulanate acid system in the case of E. coli ATCC 25922. In turn, the lowest MIC for doripenem applied alone was recorded for K. pneumoniae ATCC 31488, for which it was 0.1 µg/mL. The strain which proved to be most resistant both to doripenem and the doripenem-clavulanate acid system, was A. baumannii, with MIC of 32 µg/mL (clinical isolate) and 16 µg/mL (reference strain). Cytometric analysis for P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 showed changes in cells following exposure to limiting concentrations of the active substance. Conclusions: Analysis of MIC supplies important information concerning microbial sensitivity to active substances, mainly in terms of limiting concentrations causing mortality or vitality of the tested species, which is essential when selecting appropriate antibiotic therapy.

Dielectrophoresis System for Testing Antimicrobial Susceptibility of Gram-Negative Bacteria to ß-Lactam Antibiotics.

Gram-negative bacteria (GNBs) are common pathogens causing severe sepsis. Rapid evaluation of drug susceptibility would guide effective antibiotic treatment and promote life-saving. A total of 78 clinical isolates of 13 Gram-negative species collected between April 2013 and November 2013 from two medical centers in Tainan were tested. Bacterial morphology changes in different concentrations of antibiotics were observed under the electric field of a quadruple electrode array using light microscopy. The minimal inhibitory concentrations (MICs) of four antimicrobial agents, namely, cefazolin, ceftazidime, cefepime, and doripenem, were determined by the dielectrophoretic antimicrobial susceptibility testing (dAST) and by the conventional broth dilution testing (BDT). The antibiotics at the concentration of 1× MIC induced obvious morphological changes in susceptible GNBs, including cell elongation, cell swelling, or lysis, at 90 min. In contrast, resistant strains remained unchanged. The MIC results measured by dAST were in good agreement with those of BDT (essential agreement 95.6%). The category agreement rate was 89.2%, and the very major errors rate for dAST was 2.9%. In conclusion, dAST could accurately determine drug susceptibility within 90 min. Comprehensive tests by dAST for more drugs against more GNB species are possible in the future.